The long term goal of this research is to understand the process of segment formation in diverse animal phyla. This will contribute toward an understanding of how ancestral developmental processes were modified over the course of evolution, resulting in the dramatic differences that are observed between species. To approach this problem, the function of an engrailed-class protein (ht-en) will be examined in the leech, Helobdella triseflalis. engrailed (en) is a homeobox protein originally identified in Drosophila melanogaster. Homologues of en have since been identified in species throughout the animal kingdom, including human. In all species, en-class proteins appear to function in bCLh pattern formation and in neurogenesis. Consequently, determining the role of an en-class protein in a lower eukaryote (i.e., leech) is likely to contribute toward an understanding of normal development, and also to anomalies associated with abnormal development in higher eukaryotes (i.e., human). The function of ht-en will be examined by manipulating its expression during leech embryogenesis. Specifically, ht-en progenitor cells will be ablated using a 485 nm laser beam; ht-en expression will be abrogated with microinjected antisense oligodeoxynucleotides or ribozymes; and ht-en will be expressed ectopically in selected cell lineages. The effects of these manipulations will be measured against a previously observed fused ganglia phenotype, which results upon elimination of ht-en expressing cells during normal leech development.